1. Identify the characteristics of plasmid DNA that allow for the cloning and propagation of an exogenous DNA. (p.316)
Plasmids are small circular DNA duplexes in bacteria. They replicate independently from the host chromosome and can be modified into vectors for introduction of foreign DNA into a bacerium. Commercially available cloning vectors will contain three parts:
Plasmid origin of replication - where replication of the plasmid begins
Polylinker - Location where endonucleases can cut the plasmid and allow for insertion and annealing of foreign DNA by T4 DNA ligase.
Drug resistance gene - for the selection of successfully transformed bacteria; unsuccessful transformations will die in antibiotic agar while successful tranformations will form colonies that can be further assayed.
2. Describe the enzymatic mechanism and uses of reverse transcriptase. (p.313)
Reverse transcriptase is an RNA-dependent DNA polymerase obtained from retroviruses which have an RNA genome. It synthezies a complementry DNA (cDNA) using an mRNA template and an oligo(dT) DNA primer. Subsequent amiplication of cDNA by PCR enables cloning into a suitable vector.
3. List the DNA elements that shoud be located on an expression vector in order to express large amounts of a protein in a prokaryotic or eukaryotic host. (p.323-324)
Prokaryotes
To express copius amounts of protein in prokaryotes, the gene must be inserted in the plasmid vector downstream from a strong, inducible promotor. This allows regulation and control of bacterial expression so that they don't produce so much right away and kill themselves. Additionally, the ribosome binding site (Shine-Dalgarno site) must be placed appropriately in front of the initiation codon. Downstream of the gene of interest, a termination signal needs to be included. Lastly, eukaryotic cDNA of the protein must be used because prokaryotes lack the splicing mechanism necessary for post-translational modification.
Eukaryotes
Expression of proteins in eukaryotes are important for products of therapeutic importance, especially when specific post-translational modifications are required for protein function. Gene must be insteted downstream of a strong, inducible promoter to allow control over gene expression. Dosntream of the gene of interest, termination signals need to be included. Eukaryotic cDNA of a protein or genetic clone DNA may be used because eukaryotes have the necessary post-translational modification mechanisms to splice genomic clone DNA. However, it is necessary to choose an appropriate cell type as the host to insure the proper post-translational modifications are made.
4. Differentiate between cDNA and genomic libraries. (p.320)
Genomic Library
Contains all of the DNA from the genome of a particular cell type inserted into a collection of bacteriophage or YACs.
cDNA Library
cDNA library contains complementry DNAs from all of the mRNAs of a cell type inserted into a collection of bacteriophage.
5. List the steps involved in western blot analysis of proteins. (p.325)
Western blot is a method to study proteins by separating by gel electrophoresis into separate bands of individual proteins by size and charge. Separated bands are transfered to filter membrane and incubated with antibody that specifically reacts with protein of interest. Radioactive or chromogenic agents are then added to react with the antibody and yield a spot on an x-ray film or a color reaction to detect protein of interest.
6. List the characteristics of YAC libraries and why they are used. (p.325)
YAC (Yeast Artifical Chromosome) libraries are designed for cloning large DNA segments (>100kb). They are highly stable and maintained as a spearate chromosome in the yeast host cell. It mimics chromsomes because it contains a centromere, origin of dnA replication (autonomous replication sequence or ARS) and telomere-like sequences at the ends that maintain chromosome stability. With a selectable marker, they can selected for in yeast. YACs are used for physical mapping of human genomic DNA and analysis of large transcription units.
7. Briefly describe the steps involved in isolating a gene starting from an isolated protein. (p.311)
There are two methods to isolate a gene starting from an isolated protein.
Method 1
(1) Isolate protein on the basis of its molecular function (e.g. enzymatic or hormonal activity)
(2) Determine partial amino acid sequence of protein
(3) Synthesize oligonucleotides that correspond to portions of the amino acid sequence
(4) Use oligonucleotides as probes to select cDNA or genomic clone encoding the protein from a library
(5) Sequence isolated gene
Method 2
(1) Isolate protein on the basis of its molecular function (e.g. enzymatic or hormonal activity)
(2) Use the protein to produce specific antibodies (Ab)
(3) Use Ab as a probe to select cDNA clone encoding the protein from a library
(4) Sequence isolated gene
8. Describe the steps involved in producing transgenic animals. (p.327)
Transgenic animals can be produced by cloning and injecting a purified gene into a fertilized mouse egg pronucleus. The egg is implanted in a pseudopregnant foster mother mouse that has been treated with hormones to make it think that its is pregnant. DNA is isolated from a small piece of tail of off spring and hybridized with a probe to identify transgentic animals. Transgenic mice are bred to establish a transgenic line of mice.
9. Match the following termps with a list of their definitions:
Gene Therapy
Gene therapy is the delivery of genes to a patient in an attempt to cure a genetic or acquired disease. It is defined as the insertion of a gene (or other genetic element) into an organism which corrects a genetic dfect and can be viewed simplistically as recombinant DNA technology in a human host.
Plasmid
Plasmids are small circular DNA duplexes in a bacterial cell that replicate independently from host chromosomes. They often carry accessory genes for inactivation of antibiotics, metabolism of natural products, and production of toxins.
T4 DNA ligase
T4 DNA ligase creates a new phosphodiester bond between two DNA fragments after formation of hydrogen bonds.
Plasmid Transformation
Process of introducing a plasmid into the bacterium such that it is transformed to a new phenotype.
Origin of Replication
Start position of DNA replication.
cDNA
Complementry DNA synthesized by reverse transcriptase from mRNA transcripts.
Transgenic Animal
Animal containing foreign or modified genes
Genomic/cDNA Libraries
Genomic library contains all the DNA from the genome of a particular cell type inserted into a collection of bacteriophage or YACs. cDNA library contains complementry DNA from all of the mRNA of a cell type inserted into a collection of bacteriophage.
Expression Vector
Contains all the necessary componenents necessary for the expression of a gene of interest in prokaryotic or eukaryotic hosts. Typically, that includes a strong inducible promoter plus a ribosome binding site upstream of gene of interest, and a termination codon downstream of the genen of interest. Prokarytic and eukaryotic hosts can use cDNA of a protein as the eukaryotic gene to be expressed but only eukaryotic hosts can use genomic clones because prokaryotic cells lack the necessary post-translational modification capabilities.
Inducible Promoter
A promoter that you can easily regulate and "induce" to activate gene expression.
Western Blot
Common method of studing proteins of interest by separating proteins by size and shape through gel electrophoresis and detecting genes of interest using specific antibodies and radiologic or chromogenic secondary antibodies.
Objectives
1. Identify the characteristics of plasmid DNA that allow for the cloning and propagation of an exogenous DNA. (p.316)
Plasmids are small circular DNA duplexes in bacteria. They replicate independently from the host chromosome and can be modified into vectors for introduction of foreign DNA into a bacerium. Commercially available cloning vectors will contain three parts:
Plasmid origin of replication - where replication of the plasmid begins
Polylinker - Location where endonucleases can cut the plasmid and allow for insertion and annealing of foreign DNA by T4 DNA ligase.
Drug resistance gene - for the selection of successfully transformed bacteria; unsuccessful transformations will die in antibiotic agar while successful tranformations will form colonies that can be further assayed.
2. Describe the enzymatic mechanism and uses of reverse transcriptase. (p.313)
Reverse transcriptase is an RNA-dependent DNA polymerase obtained from retroviruses which have an RNA genome. It synthezies a complementry DNA (cDNA) using an mRNA template and an oligo(dT) DNA primer. Subsequent amiplication of cDNA by PCR enables cloning into a suitable vector.
3. List the DNA elements that shoud be located on an expression vector in order to express large amounts of a protein in a prokaryotic or eukaryotic host. (p.323-324)
Prokaryotes
To express copius amounts of protein in prokaryotes, the gene must be inserted in the plasmid vector downstream from a strong, inducible promotor. This allows regulation and control of bacterial expression so that they don't produce so much right away and kill themselves. Additionally, the ribosome binding site (Shine-Dalgarno site) must be placed appropriately in front of the initiation codon. Downstream of the gene of interest, a termination signal needs to be included. Lastly, eukaryotic cDNA of the protein must be used because prokaryotes lack the splicing mechanism necessary for post-translational modification.
Eukaryotes
Expression of proteins in eukaryotes are important for products of therapeutic importance, especially when specific post-translational modifications are required for protein function. Gene must be insteted downstream of a strong, inducible promoter to allow control over gene expression. Dosntream of the gene of interest, termination signals need to be included. Eukaryotic cDNA of a protein or genetic clone DNA may be used because eukaryotes have the necessary post-translational modification mechanisms to splice genomic clone DNA. However, it is necessary to choose an appropriate cell type as the host to insure the proper post-translational modifications are made.
4. Differentiate between cDNA and genomic libraries. (p.320)
Genomic Library
Contains all of the DNA from the genome of a particular cell type inserted into a collection of bacteriophage or YACs.
cDNA Library
cDNA library contains complementry DNAs from all of the mRNAs of a cell type inserted into a collection of bacteriophage.
5. List the steps involved in western blot analysis of proteins. (p.325)
Western blot is a method to study proteins by separating by gel electrophoresis into separate bands of individual proteins by size and charge. Separated bands are transfered to filter membrane and incubated with antibody that specifically reacts with protein of interest. Radioactive or chromogenic agents are then added to react with the antibody and yield a spot on an x-ray film or a color reaction to detect protein of interest.
6. List the characteristics of YAC libraries and why they are used. (p.325)
YAC (Yeast Artifical Chromosome) libraries are designed for cloning large DNA segments (>100kb). They are highly stable and maintained as a spearate chromosome in the yeast host cell. It mimics chromsomes because it contains a centromere, origin of dnA replication (autonomous replication sequence or ARS) and telomere-like sequences at the ends that maintain chromosome stability. With a selectable marker, they can selected for in yeast. YACs are used for physical mapping of human genomic DNA and analysis of large transcription units.
7. Briefly describe the steps involved in isolating a gene starting from an isolated protein. (p.311)
There are two methods to isolate a gene starting from an isolated protein.
Method 1
(1) Isolate protein on the basis of its molecular function (e.g. enzymatic or hormonal activity)
(2) Determine partial amino acid sequence of protein
(3) Synthesize oligonucleotides that correspond to portions of the amino acid sequence
(4) Use oligonucleotides as probes to select cDNA or genomic clone encoding the protein from a library
(5) Sequence isolated gene
Method 2
(1) Isolate protein on the basis of its molecular function (e.g. enzymatic or hormonal activity)
(2) Use the protein to produce specific antibodies (Ab)
(3) Use Ab as a probe to select cDNA clone encoding the protein from a library
(4) Sequence isolated gene
8. Describe the steps involved in producing transgenic animals. (p.327)
Transgenic animals can be produced by cloning and injecting a purified gene into a fertilized mouse egg pronucleus. The egg is implanted in a pseudopregnant foster mother mouse that has been treated with hormones to make it think that its is pregnant. DNA is isolated from a small piece of tail of off spring and hybridized with a probe to identify transgentic animals. Transgenic mice are bred to establish a transgenic line of mice.
9. Match the following termps with a list of their definitions:
Gene Therapy
Gene therapy is the delivery of genes to a patient in an attempt to cure a genetic or acquired disease. It is defined as the insertion of a gene (or other genetic element) into an organism which corrects a genetic dfect and can be viewed simplistically as recombinant DNA technology in a human host.
Plasmid
Plasmids are small circular DNA duplexes in a bacterial cell that replicate independently from host chromosomes. They often carry accessory genes for inactivation of antibiotics, metabolism of natural products, and production of toxins.
T4 DNA ligase
T4 DNA ligase creates a new phosphodiester bond between two DNA fragments after formation of hydrogen bonds.
Plasmid Transformation
Process of introducing a plasmid into the bacterium such that it is transformed to a new phenotype.
Origin of Replication
Start position of DNA replication.
cDNA
Complementry DNA synthesized by reverse transcriptase from mRNA transcripts.
Transgenic Animal
Animal containing foreign or modified genes
Genomic/cDNA Libraries
Genomic library contains all the DNA from the genome of a particular cell type inserted into a collection of bacteriophage or YACs. cDNA library contains complementry DNA from all of the mRNA of a cell type inserted into a collection of bacteriophage.
Expression Vector
Contains all the necessary componenents necessary for the expression of a gene of interest in prokaryotic or eukaryotic hosts. Typically, that includes a strong inducible promoter plus a ribosome binding site upstream of gene of interest, and a termination codon downstream of the genen of interest. Prokarytic and eukaryotic hosts can use cDNA of a protein as the eukaryotic gene to be expressed but only eukaryotic hosts can use genomic clones because prokaryotic cells lack the necessary post-translational modification capabilities.
Inducible Promoter
A promoter that you can easily regulate and "induce" to activate gene expression.
Western Blot
Common method of studing proteins of interest by separating proteins by size and shape through gel electrophoresis and detecting genes of interest using specific antibodies and radiologic or chromogenic secondary antibodies.